Not applicable.
Not applicable.
The field of this invention is the production of recombinant proteins, in particular, using regulated gene expression (the xynA promoter) in Aureobasidium pullulans. 
The yeast-like fungus Aureobasidium pullulans has been isolated from various ecosystems, and different isolates display morphologic diversity. The fungus has been extensively studied for its capability to produce high levels of xylanase and pullulan and for its ubiquitous distribution on plant leaves. A. pullulans Y-2311-1has been shown to produce the highest levels of xylanase among several xylanolytic fungi [Leathers, T. D. (1986) Appl. Environ. Microbiol. 52:1026-1030; Leathers et al. (1984) Biotechnol. Bioeng. Symp. 14:225-250; Leathers et al. (1986) Biotechnol. Lett. 8:867-872]. The fungus is able to grow on glucose, xylose, glycerol, or arabinose as carbon source. Plant biomass such as wheat bran, corn fiber and starch (but not pure cellulose) also supports rapid growth of the fungus. Xylan and xylose induce while glucose represses the production of xylanolytic activity. Four xylanases which differ in isoelectric points are produced under inducing conditions, but most of the activity is attributed to two xylanase molecules (20 and 25 kDa) [Leathers, T. D. (1986) supra; Li et al. (1993) Appl. Environ. Microbiol. 59:3212-3218], differing in glycosylation, encoded by a single gene (xynA) in the fungal genome [Li and Ljungdahl (1994) Appl. Environ. Microbiol. 60:3160-3166; Li and Ljungdahl (1997) U.S. Pat. No. 5,591,619]. The two xylanases account for over 80% by weight of the total extracellular proteins in cultures grown on arabinoxylan-rich or xylose medium. It has been shown that either xylan or xylose induced the transcription of the xylanase gene [Li and Ljungdahl (1994) supra; Vanden Wymelenberg et al. (1999) FEMS Microbiol. Lett. 181:205-205]. The genomic xynA was amplified by polymerase chain reaction (PCR), and sequencing analysis revealed that a 59-bp intron was located in the DNA region encoding the signal peptide. The xylanase cDNA has been inserted into a plasmid under the control of a yeast Gall promoter and high levels of xylanase were secreted by Saccharomyces cerevisiae[Li and Ljungdahl (1996) Appl. Environ. Microbiol. 62:209-213; Li and Ljungdahl (1997) supra].
Hemicellulose, second only to cellulose in abundance on earth, comprises xylan as the main constituent. Xylan is a hetero-polymer comprising beta- 1,4-linked xylose units as a backbone and side chains which contain pentose, hexose, and acetyl groups. The pentose (arabinose) is esterified to free and lignin phenolic (feruloyl and p-coumaroyl) groups [Christov and Prior (1993) Enzyme Microb. Technol. 15:460-475]. Xylan can be readily converted to xylose and other monomeric sugars through either chemical or enzymatic hydrolysis of agricultural and forestry waste biomass. Enzymatic degradation of hemicelluloses requires the participation of several enzymes including xylanase (EC3.2.1.8), xcex2-xylosidase (EC3.2.1.37), xcex1-L-arabinofuranosidase (EC3.2.1.55), xcex1-glucuronidase (EC3.2.1.1), acetyl xylan esterase (EC3.1.1.6) as well as p-coumaroyl and feruloyl esterases [Borneman et al. (1993) In: Hemicellulose and Hemicellulases, M. P. Coughlan and G. Hazlewood (ed.), Portland Press, Cambridge, UK, pp. 85-102; Christov and Prior (1993) supra]. Due to efficient conversion of agricultural plant residues to xylose and widespread use of xylitol, an alcohol derived from xylose in food products, using xylose as an inducer in large-scale fermentation has become cost effective in comparison to other commonly used inducers for fermentation.
There is a long-felt need in the art for regulatable promoters for use in recombinant protein production, where those promoters are regulated and recombinant proteins can be produced using inexpensive substrates.
It is an object of the present invention to provide a regulated promoter and a signal peptide (and the amino acid sequence encoding it) as well as methods for using the promoter/signal peptide coding sequence for efficient and economical secreted expression of a protein of interest in a eukaryotic cell, especially a fungal cell, for example, Aureobasidium pullulans). The signal peptide of the present invention is the XynA (also called APX-II) signal peptide. In general, proteins secreted from cells are synthesized as precursor molecules containing hydrophobic N-terminal signal peptides. The signal peptides direct transport of the protein to its target membrane and are then cleaved by a peptidase on the membrane, such as the endoplasmic reticulum, when the protein passes through it.
This invention also provides for genomic DNA sequence of the xynA locus of A. pullulans and the associated transcription regulatory sequences (and the sequences encoding the XynA signal peptide). The gene encoding both these peptides is termed xynA herein. The DNA sequence of the gene as it occurs in A. pullulans is given in Table 1 and in SEQ ID NO:1. The transcription start site is about nucleotide 678, based on the size of the xynA transcript, and the coding sequence for the XynA precursor protein is from nucleotide 788 to 1469, excluding a translation stop codon and excluding the intron from nucleotides 821 to 880. There is an intron which extends from nucleotide 821 to 880 (within the coding sequence for the signal peptide), as shown in Table 1, and in SEQ ID NO:1. The DNA sequence provided herein including the intron is useful for recombinantly expressing a secreted protein via the APX-II signal peptide in Aureobasidium species and other host species capable of splicing out the intron. The xynA regulated promoter without the intron is useful for recombinantly expressing the XynA mature protein in S. cerevisiae or other host species which are not capable of splicing out the intron. The regulatable xynA promoter is useful for the regulated expression of an operably linked coding sequence, with high levels of gene expression obtained in the presence of the inducing substrates xylan or xylose, where the medium contains glucose a concentration less than about 0.02% (w/v). The xynA xylan- or xylose-responsiveness transcription regulatory sequence is embodied in the nucleotide sequence as set forth in SEQ ID NO:1, or fragments thereof sufficient to retain xylan- or xylan-responsiveness and promoter activity, preferably fragments corresponding to nucleotides 478 to 678, 378 to 678, 278 to 678, 178 to 678, 78 to 678, 1 to 678, and 1 to 733, all as set forth in SEQ ID NO:1.
The XynA signal peptide of this invention has a sequence as given in SEQ ID NO:2 from amino acid 1 to amino acid 34. The term xe2x80x9cAPX-II signal sequencexe2x80x9d encompasses not only the exact sequence given, but also equivalent sequences which have additions, substitutions or deletions which do not interfere with the function of the signal peptide. The coding region for the XynA signal peptide in a construct 5xe2x80x2 to a coding sequence for a protein of interest functions to produce an expression product which is secreted efficiently and with high relative purity in the extracellular medium of a fungus, especially A. pullulans. The skilled worker, following the teachings herein, is enabled to make changes to the signal sequence which do not adversely affect its function and thus is enabled to make a large number of operative embodiments of this signal peptide.
The present invention further provides a method for the regulated expression of a protein of interest in a recombinant cell of the genus Aureobasidium, for example, Aureobasidium pullulans, using the xynA promoter derived from A. pullulans operably linked to DNA encoding the protein of interest. The nucleotide sequence of the preferred embodiment of the xynA promoter is given in SEQ ID NO:1, nucleotides 1 to 733.
Expression of the protein of interest is induced when xylose or xylan is added to culture medium having a glucose concentration less than 0.02% (w/v). Desirably xylose is added to a final concentration from about 0.05% (w/v) to about 5.0% (w/v), advantageously from about 0.5% (w/v) to about 1.25% (w/v), and as exemplified, 1% (w/v) xylose. Where xylan is used, the concentration of xylan in the medium is desirably from about 0.05% to about 5.0% (w/v).